The present invention, in some embodiments thereof, relates to novel glycogen synthase kinase-3 (GSK-3) inhibitors and, more particularly, but not exclusively, to novel selective inhibitors of glycogen synthase kinase-3 (GSK-3) and to the use of such inhibitors in, for example, the treatment of biological conditions associated with GSK-3 activity.
Protein kinases and phosphorylation cascades are essential for life and play key roles in the regulation of many cellular processes including cell proliferation, cell cycle progression, metabolic homeostasis, transcriptional activation and development. Aberrant regulation of protein phosphorylation underlies many human diseases, and this has prompted the development and design of protein kinase inhibitors. Most of the protein kinase inhibitors developed so far compete with ATP for its binding site. These inhibitors, although often very effective, generally show limited specificity due to the fact that the ATP binding site is highly conserved among protein kinases.
Other sites, such as the substrate's binding site, show more variability in their shape and amino acid compositions and may serve as favorable sites for drug design. Understanding of substrate recognition and specificity is thus essential for development of substrate competitive inhibitors. This knowledge, however, is limited by the scarce amount of structural data regarding substrate binding.
Glycogen synthase kinase-3 (GSK-3) is a constitutively active serine/threonine kinase that modulates diverse cellular functions including metabolism, cell survival and migration, neuronal signaling and embryonic development. Deregulation of GSK-3 activity has been implicated in the pathogenesis of human diseases such as, for example, type-2 diabetes, neurodegenerative disorders and psychiatric disorders. Selective inhibition of GSK-3 is thought to be of therapeutic value in treating these disorders [Bhat et al. (2004). J. Neurochem. 89, 1313-7; Cohen, P. & Goedert, M. (2004). Nat. Rev. Drug Discov. 3, 479-87; Meijer et al. (2004) Trends Pharmacol Sci 25, 471-80; Eldar-Finkelman et al. Biochim Biophys Acta 1804, 598-603; Martinez, A. & Perez, D. I. (2008) J. Alzheimers Dis. 15, 181-91].
Recently, it has been found that GSK-3 is also involved in the pathogenesis of cardiovascular diseases [Cheng et al. 2010 J. Mol Cell Cardiol, in press; Kerkela et al. 2008, J. Clin. Invest. 118:3609-18], of malaria and trypanosomiasis [Droucheau et al. 2004, BBRC, 1700:139-140; Ojo et al. 2008, Antimicrob Agents Chemother, 37107-3717], and in stem cell maintenance or differentiation [Wray et al. 2010 Biochem Soc Trans 1027-32].
In view of the wide implication of GSK-3 in various signaling pathways, development of specific inhibitors for GSK-3 is considered both promising and important regarding various therapeutic interventions as well as basic research.
Some mood stabilizers were found to inhibit GSK-3. However, while the inhibition of GSK-3 both by lithium chloride (LiCl) (WO 97/41854) and by purine inhibitors (WO 98/16528) has been reported, these inhibitors are not specific for GSK-3. In fact, it was shown that these drugs affect multiple signaling pathways, and inhibit other cellular targets, such as inositol monophosphatase (IMpase) and histone deacetylases.
Similarly, an engineered cAMP response element binding protein (CREB), a known substrate of GSK-3, has been described (Fiol et al, 1994), along with other potential GSK-3 peptide inhibitors (Fiol et al, 1990). However, these substrates also only minimally inhibit GSK-3 activity.
Other GSK-3 inhibitors have been reported. Two structurally related small molecules SB-216763 and SB-415286 (GlaxoSmithKline Pharmaceutical) that specifically inhibited GSK-3 were developed and were shown to modulate glycogen metabolism and gene transcription as well as to protect against neuronal death induced by reduction in PI3 kinase activity (Cross et al., 2001; Coghlan et al., 2000). Another study indicated that Induribin, the active ingredient of the traditional Chinese medicine for chronic myelocytic leukemia, is a GSK-3 inhibitor. However, Indirubin also inhibits cyclic-dependent protein kinase-2 (CDK-2) (Damiens et al., 2001). These GSK-3 inhibitors are ATP competitive and were identified by high throughput screening of chemical libraries. It is generally accepted that a major drawback of ATP-competitive inhibitors is their limited specificity (see, for example, Davies et al., 2000).
Some of the present inventors have previously reported of a novel class of substrate competitive inhibitors for GSK-3 [Plotkin et al. (2003) J. Pharmacol. Exp. Ther., 974-980], designed based on the unique substrate-recognition motif of GSK-3 that includes a phosphorylated residue (usually serine) in the context of SXXXS(p) (where S is the target serine, S(p) is phosphorylate serine and X is any amino acid) [see also Woodgett & Cohen (1984) Biochim. Biophys. Acta. 788, 339-47; Fiol et al. (1987) J. Biol. Chem. 262, 14042-8]. Structural studies of GSK-3β identified a likely docking site for the phosphorylated residue; it is a positively charged binding pocket composed of Arg96, Arg180, and Lys205 [Dajani et al. (2001) Cell 105, 721-32; ter Haar et al. (2001) Nature Structural Biology 8, 593-6].
The short phosphorylated peptides patterned after the GSK-3 substrates behaved as substrate competitive inhibitors (Plotkin et al., 2003, supra), with the L803 peptide, KEAPPAPPQS(p)P (SEQ ID NO:4), derived from the substrate heat shock factor-1 (HSF-1) showing the best inhibition activity of those evaluated. An advanced version of L803, the cell permeable peptide L803-mts, was shown to promote beneficial biological activities in conditions associated with diabetes, neuron growth and survival, and mood behavior [Kaidanovich-Beilin & Eldar-Finkelman (2005) J. Pharmacol. Exp. Ther. 316:17-24; Rao et al. (2007) Diabetologia 50, 452-60; Kim et al. (2006) Neuron 52, 981-96; Chen et al. (2004) Faseb J 18, 1162-4; Kaidanovich-Beilin et al. (2004) Biol. Psychiatry. 55:781-4; Shapira et al. (2007) Mol. Cell Neurosci. 34, 571-7].
While further focusing on substrate recognition of GSK-3, three positions in the vicinity of the catalytic site (Phe67 in the P-loop, Gln89 and Asn95) were identified as important for GSK-3 substrates binding [Ilouz et al. (2006) J. Biol. Chem. 281, 30621-30], and a cavity bordered by loop 89-QDKRFKN-95 (SEQ ID NO:2) located in the vicinity of the GSK-3β catalytic core, has also been identified as a substrate binding subsite.
In-silico modeling of the interaction of GSK-3 with its substrates pCREB and p9CREB, corroborated by mutation experiments, suggested that the substrates bind in the deep trough between the N- and C-terminal lobes of the kinase, as illustrated in Background Art FIG. 1 (Ilouz et al. 2006, supra). It was further suggested that the pre-phosphorylated S133p residue is located in the phosphate binding pocket of GSK-3 formed by residues R96, R180 and K205, and the phosphorylation target S129 points toward they phosphate of ATP.
WO 2012/101599 describes further studies conducted for identifying sites of GSK-3 that play an important role in binding GSK-3 substrates. In these studies, a role of Phe93, as well as of other amino acids within the 89-95 loop of a GSK-3 enzyme, in interacting with GSK-3 substrates and hence with GSK-3 substrate competitive inhibitors, was uncovered, thereby indicating that a putative substrate competitive inhibitor should exhibit an interaction with the Phe93 residue, or with an equivalent amino acid thereof, in a GSK-3 enzyme Peptidic substrate competitive GSK-3 inhibitors were designed after the recognition motif of HSF while modifying the peptide's hydrophobic nature by replacing hydrophilic polar amino acids by hydrophobic amino acids residues such as alanine and proline. Exemplary such substrate competitive inhibitors, which feature a hydrophobic amino acid residue at the first position upstream the phosphorylated serine or threonine residue, exhibited improved activity compared to, for example, L803.
WO 2012/101601 describes additional studies in which initial models obtained by rigid body docking of selected L803 conformers to GSK-3β with the geometric-electrostatic-hydrophobic version of MolFit followed by filtering based on statistical propensity measures and solvation energy estimates, were subjected to molecular dynamics (MD) simulations. These computations provided further understanding on the binding of the inhibitor. The computational model structures, supported by experimental data, have shown that a modified L803 peptide, which features a Phe residue at the C-terminus of L803, and which is termed L803F, exhibits a substantially improved interaction with Phe 93, via its Pro8 and Phe12, and with the hydrophobic surface patch of GSK-3β.
Further in silico modeling suggested that while GSK-3 substrates interact with the positive pocket delimited by residues R96, R180 and K205, with the substrate binding cavity delimited by the P-loop and loop 89-95, with the protruding F93 residue, and with the hydrophobic surface patch opposite the P-loop, which consists of residues V214, Y216 and I217, the inhibitor L803 uses only part of the sub-sites mentioned above: the pre-phosphorylated serine binds in the positive pocket but the other contacts are hydrophobic (Licht-Murava et al., J. Mol. Biol. (2011) 408, 366-378). Thus, L803 interacts with GSK-3 F93 and with the hydrophobic patch but it does not interact with the P-loop or with the substrate binding cavity.
Additional background art includes U.S. Pat. Nos. 6,780,625 and 7,378,432; WO 2004/052404 and WO 2005/000192; WO 01/49709; Liberman, Z. & Eldar-Finkelman, H. (2005) J. Biol. Chem. 280, 4422-8; Liberman et al. (2008) Am. J. Physiol. Endocrinol. Metab. 294, E1169-77; Bertrand et al. (2003) J. Mol. Biol. 333, 393-407; and Palomo et al. J. Med. Chem. (2012) Feb. 23; 55(4):1645-61.